1. Field of the Invention
The invention in the fields of microbiology and medicine relates to methods for rapid early detection of mycobacterial disease in humans based on the presence of antibodies to particular “early” mycobacterial protein antigens, and reactive epitopes thereof, which have not been previously recognized for this purpose. Assay of such antibodies on selected mycobacterial proteins, peptides thereof, or fusion polypeptides (peptide multimers, polyproteins) permits diagnosis of TB earlier than has been heretofore possible. Also provided is a surrogate marker for screening populations at risk for TB, in particular subjects infected with human immunodeficiency virus (HIV).
2. Description of the Background Art
Estimates by the World Health Organization (WHO) in 1995 suggested that approximately 90 million new cases of tuberculosis (“TB”) will occur during the coming decade leading to about 30 million deaths (Raviglione, M C et al., 1995, JAMA. 273:220-226). The spread of HIV in populations already having a high incidence of TB has resulted in a resurgence of TB all over the world (Raviglione, M C et al., 1992, Bull WHO 70:515-526; Harries A. D., 1990, Lancet. 335:387-390) and has stimulated renewed interest in improved vaccines, diagnostics, drugs and drug delivery regimens for TB. Furthermore, the immune dysfunction caused by HIV infection leads to a high rate of reactivation of latent TB, increased susceptibility to primary disease, as well as an accelerated course of disease progression (Raviglione et al., 1992, supra; 1995, supra; Shafer R W et al., 1996, Clin. Infect. Dis. 22:683-704; Barnes P F et al., 1991, N. Engl. J. Med. 324:1644-1650; Selwyn P A et al., 1989, N. Engl. J. Med. 320:545-550).
The importance of cellular immunity for protection against TB is well established. Much of the work in this field is focused on defining the antigens of the causative bacterium, Mycobacterium tuberculosis (M. tuberculosis; also abbreviated herein as “Mtb”) that can elicit effective immunity and on understanding the role of various cell populations in host-pathogen interactions (Andersen, P et al., 1992, Scand. J. Immunol. 36:823-831; Havlir, D V et al., 1991, Infect. Immun. 59:665-670; Orme, I M et al., 1993, J. Infect. Dis. 167:1481-1497).
Delayed hypersensitivity measured as cutaneous immune reactivity to a purified protein derivative of Mtb (abbreviated “PPD”) has been the only accepted marker available for detection of latent infection with Mtb. However, the sensitivity of the PPD skin test is substantially reduced during HIV infection (Raviglione et al., 1992, supra, 1995, supra; Graham N M H et al., 1991, JAMA 267:369-373; Huebner R E et al., 1994, Clin. Infect. Dis. 19:26-32; Huebner R E et al., 1992, JAMA 267:409-410; Caiaffa W T et al., 1995, Arch. Intern. Med. 155:2111-2117). Furthermore, vaccination with a closely related mycobacterium Bacillus Calmette-Guerin (BCG) or previous exposure to other mycobacterial species can lead to false positive results in a PPD skin test. Not only does PPD reactivity fail to distinguish active, subclinical disease from latent infection, but the time between a positive skin test and development of clinical disease may range from months to several years (Selwyn P A et al., supra).
Because of the susceptibility of immunocompromised individuals to TB, the U.S. Centers for Disease Control and Prevention has recommended preventive isoniazid therapy for all HIV seropositive (HIV+), PPD-positive (PPD+) individuals. However, the optimal time for such therapy is not clear and, ideally, should coincide with replication of previously latent bacteria. Unnecessary therapy must be minimized because prolonged isoniazid treatment can have serious toxic side effects (Shafer et al., supra). The impact of such treatment on emergence of drug resistant bacteria is still unclear. The use of preventive therapy in developing countries is seriously limited by the high frequency of PPD+ individuals coupled with the lack of adequate medico-social infrastructure and economic resources. High risk populations are also found in the United States, primarily intravenous drug users, homeless people, prison inmates and residents of slum areas (Fitzgerald, J M et al., 1991, Chest 100:191-200; Graham et al., supra; Friedman, L N et al., 1996, New Engl. J. Med. 334:828-833) as well as household contacts of TB patients. Thus, discovery of additional surrogate markers for early detection and prompt treatment of active, subclinical TB in such high risk populations is urgently required.
Antibody responses in TB have been studied for several decades primarily for the purpose of developing serodiagnostic assays. Although some seroreactive antigens/epitopes have been identified, interest in antibody responses to Mtb has waned because of the lack of progress in simple detection of corresponding antibodies. Studies using crude antigen preparations revealed that healthy individuals possess antibodies that cross-react with several mycobacterial antigens presumably elicited by exposure to commensal and environmental bacteria and vaccinations (Bardana, E J et al., 1973, Clin. Exp. Immunol. 13:65-77; Das, S et al., 1992, Clin. Exp. Immunol. 89:402-406; Del Giudice, G et al., 1993, J. Immunol. 150:2025-2032; Grange, J M, 1984, Adv. Tuberc. Res. 21:1-78; Havlir, D V et al., supra; Ivanyi, J et al., 1989, Brit. Med. Bull. 44:635-649; Verbon, A et al., 1990, J. Gen. Microbiol. 136:955-964). Several mycobacterial antigens have been isolated and characterized (Young, D B et al., 1992, Mol. Microbiol. 6:133-145), including the 71 kDa DnaK, 65 kDa GroEL, 47 kDa elongation factor tu, 44 kDa PstA homologue, 40 kDa L-alanine dehydrogenase, 38 kDa PhoS, 23 kDa superoxide dismutase, 23 kDa outer membrane protein, 12 kDa thioredoxin, and the 14 kDa GroES. A majority of these antigens bear significant homology to the analogous proteins in other mycobacteria and non-mycobacterial prokaryotes (Andersen, A B et al., 1992, Infect. Immun. 60:2317-2323; Andersen, A B et al., 1989, Infect. Immun. 57:2481-2488; Braibant, M et al., 1994, Infect. Immun. 62:849-854; Carlin, N et al., 1992, Infect. Immun. 60:3136-3142; Garsia, R J et al., 1989, Infect. Immun. 57:204-212; Hirschfield, G R et al., 1990, J. Bacteriol. 172:1005-1013; Shinnick, T M et al., 1989, Nucl. Acids Res. 17:1254; Shinnick, T M et al., 1988, Infect. Immun. 56:446-451; Wieles, B et al., 1995, Infect. Immun. 63:4946-4948; Young, D B et al., supra; Zhang, Y et al., 1991, Mol. Microbiol. 5:381-391). Thus, almost all individuals (healthy or diseased) have antibodies to epitopes of conserved regions of these antigens. These antibodies are responsible for the uninformative (and possibly misleading) cross-reactivity observed with crude Mtb antigen preparations (Davenport, M P et al., 1992, Infect. Immun. 60:1170-1177; Grandia, A A et al., 1991, Immunobiol. 182:127-134; Meeker, H C et al., 1989, Infect. Immun. 57:3689-3694; Thole, J et al., 1987, Infect. Immun. 55:1466-1475).
Because such cross-reactive antibodies would mask the presence of antibodies specific for Mtb antigens, some of the purified antigens such as the 38 kDa PhoS, the 30/31 kDa “antigen 85” (discussed in more detail below), 19 kDa lipoprotein, 14 kDa GroES and lipoarabinomannan have been prepared and tested (Daniel, T et al., 1985 Chest. 88:388-392; Drowart, L et al., 1991, Chest. 100:685-687; Jackett, P S et al., 1988, J. Clin. Microbiol. 26:2313-2318; Ma, Y et al., 1986, Am Rev Respir Dis 134:1273-1275; Sada, E et al., 1990, J. Clin. Microbiol. 28:2587-2590; Sada, E D et al., 1990, J. Infect. Dis. 162:928-931; Van Vooren, J P et al., 1991, J. Clin. Microbiol. 29:2348-2350). It is noteworthy that the choice of which antigen to test was dictated primarily by (a) its availability, (b) its immunodominance in animal immunizations, or (c) ease of its biochemical purification. None of these criteria take into account the reactivity of the antigen which occurs naturally in the human immune response to mycobacterial diseases. For a time, use of the 38 kDa antigen provided the highest serological sensitivity and specificity (Daniel, T M et al., 1987, Am Rev Respir Dis 135:1137-1151; Harboe, M et al., 1992, J. Infect. Dis. 166:874-884; Ivanyi, J et al., 1989, supra). However, in contrast to antibodies against the antigens discovered by the present inventors, the presence of anti-38 kDa antibodies is associated primarily with treated, advanced and recurrent TB (Bothamley, G H et al., 1992, Thorax. 47:270-275; Daniel et al., supra Ma et al., supra.
One convention in mycobacterial protein nomenclature is the use of MPB and MPT numbers. MPB denotes a protein purified from M. bovis BCG followed by a number denoting its relative mobility in 7.7% polyacrylamide gels at a pH of 9.5. MPT denotes a protein isolated from Mtb. In proteins examined prior to this invention, no differences in the N-terminal amino acid sequence were shown between these two mycobacterial species.
Wiker and colleagues have studied a family of secreted Mtb proteins which include a complex of 3 proteins termed antigens 85A, 85B and 85C (also known as the “85 complex” or “85cx”) (Wiker, H. G. et al., 1992, Scand. J. Immunol. 36:307-319; Wiker, H. G. et al., 1992, Microbiol. Rev. 56:648-661). The corresponding components of Mtb are also actively secreted. The 85 complex is considered the major secreted protein constituent of mycobacterial culture fluids though it is also found in association with the bacterial surface. In most SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analyses, 85A and 85C are not properly resolved, whereas isoelectric focusing resolves three distinct bands.
Genes encoding six of the secreted proteins: 85A, 85B, 85C, “antigen 78” (usually referred to as the 38 kDa protein), MPB64 and MPB70 have been cloned. Three separate genes located at separate sites in the mycobacterial genome encode 85A, B and C (Content, J. et al., 1991, Infect. Immun. 59:3205-3212). A gene encoding the antigen known as MPT-32 (reported as a 45/47 kDa secreted antigen complex) has been cloned, sequenced and expressed (Laqueyrerie, A. et al., 1995, Infec. Immun. 63:4003-4010) and designated as the apa gene. The need continues for further elucidation of the biochemistry and immunochemistry of Mtb proteins and glycoproteins which are potentially important as serodiagnostic tools.
The following list shows the molecular masses of the individual components of antigen 85 complex plus two additional antigens (in SDS-PAGE) as described by Wiker and colleagues, along with alternative nomenclatures:
Ag85A=MPT44=31 kDaAg85B=MPT59=30 kDaAg85C=MPT45=31.5 kDaMPT64=26 kDaMPT51=27 kDaAg78—=38 kDaMPT32=45/47 kDa (found to be 38/42 kDa by thepresent inventors)
Wiker's group studied cross-reactions between five actively secreted Mtb proteins by crossed immunoelectrophoresis, SDS-PAGE with immunoblotting and enzyme immunoassay (EIA) using (1) polyclonal rabbit antisera to the purified proteins and (2) a mouse monoclonal antibody (“mAb”). The mAb HBT4 reacted with the MPT51 protein.
The aligned amino acid sequences listed below illustrate the homology of a fragment of 85A, 85B, 85C, 1 and MPT64. The numbers at the top correspond to the part of the sequence shown. The N-terminal sequences were determined on isolated proteins and aligned by visual inspection. The sequence from position 66 to 91 of MPT64 is the sequence deduced from the cloned gene.
SEQ1   5    10    15    20   25   30    35ID NO85A(1-39)FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGANSP ALYLL1 85B(1-39)FSRPGLPVEYLQVPS PSMGRDIKVQFQSGGNNSP AVYLL2 85C(1-37)FSRPGLPVEYLQVPSA SMGRDIKVQFQGGG   PHAVYLL3 MPT51(1-32)     APYENLMVPS PSMGRDIPVAFLAGG   PHAVYLL4 MPT64(66-91)     APYE LNITSATYQS     AIPPRG   TQAVVL5The N-terminal sequence of MPT51 showed 72% homology with the sequence of the Ag 85 components (when P at position 2 is aligned with P at position 7 of the three Ag 85 components.
Studies of TB patients showed that assays of antibodies to the Ag 85 complex had a sensitivity of about 50%. With regard to specificity, the Ag 85 components are highly cross-reactive so that positive responses are expected (and found) in healthy controls, particularly in geographic areas of high exposure to atypical mycobacteria. The different degree of specificity is thus highly dependent on the kind of control subjects used. It is noteworthy that traditional BCG vaccination does not appear to induce a significant antibody response, though it is interesting that antibodies to mycobacterial antigens increased when anti-TB chemotherapy was initiated. A number of studies have examined antibodies to various Mtb antigen in TB sera or sera of patients with other diseases. See, for example, Espitia, C et al., 1989, Clin Exp Immunol 77:373-377; Van Vooren, J P et al., 1991, J. Clin. Microbiol. 29:2348-2350; Wiker et al. (supra). C. Espitia et al., 1995, Infect. Immun. 63:580-584, found reciprocal cross-reactivity between a Mtb 50/55 kDa protein and a M. bovis BCG 45/47 kDa antigen using a rabbit polyclonal antiserum against the M. bovis protein and a mAb against the Mtb antigen. Both antigens were secreted glycoproteins. The N-terminal sequences and total amino acid content of these proteins were very similar. 2D gel electrophoresis showed at least seven different components in the Mtb 50/55 kDa antigen. In solid-phase immunoassays, purified Mtb 50/55 kDa protein was recognized by sera from 70% of individuals (n=77) with pulmonary TB. The N-terminus of the Mtb 41 kDa antigen known as MPT32 was very similar to the N-termini of the 50/55 kDa- and the 45-47 kDa proteins. The authors speculated about a diagnostic potential for these antigens based on these observation However, the potential of this antigen as an early diagnostic agent for TB was neither analyzed nor even suggested.
Importantly, there has been a deficiency in the art of analysis of antibodies at different stages of disease, which is one of the primary objectives addressed by this invention. None of the antigens studied so far, with the possible exception of MPT32 (as will be described herein) has emerged as a suitable candidate for development of a diagnostic assay for early stages of TB. Since antigens/epitopes recognized during natural infection and disease progression in humans may differ substantially from those recognized by animals upon artificial immunization (Bothamley, G. et al., 1988, Eur. J. Clin. Microbiol. Infect. Dis. 7:639-645; Calle, J. et al., 1992, J. Immunol. 149:2695-2701; Hartskeerl, R. A. et al., 1990, Infect. Immun. 58:2821-2827; Laal, S. et al., 1991, Proc. Natl. Acad. Sci. USA. 88:1054-1058; Meeker, H. C. et al., 1989, Infect. Immun. 57:3689-3694; Verbon, A., 1994, Trop. Geog. Med. 46:275-279), there is a pressing need in the art for selection of antigens based on their ability to stimulate the human immune system. This would permit the identification of useful protein antigens and peptide epitopes for use in the design of diagnostic assays for early detection of TB and for vaccines.
TB in HIV Infected Subjects
Although the literature on TB infection in subjects not infected with HIV is extensive, reports on antibody responses of HIV/TB patients to Mtb, have been scant and controversial. Farber, C. et al., 1990, J. Infect. Dis, 162:279-280, reported the presence of antibodies to the p32 antigen (same as 85A) in 7 of 8 HIV/TB patients. Da Costa, C. et al., 1993, Clin. Exp. Immunol. 91:25-29, reported the presence of anti-lipoarabinomannan (LAM) antibodies in 35% of such patients. Barer, L. et al., 1992, Tuber. Lung. Dis. 73:187-191, reported anti-PPD antibodies in 36% of HIV/TB patients. Martin-Casabona, N. et al., 1992, J. Clin. Microbiol. 30:1089-1093, reported anti-sulfolipid (SLIV) antibodies in 73% of their patients. In addition, van Vooren, P. et al., 1988, Tubercle. 69:303-305, reported that anti-p32 antibodies were detectable in an HIV/TB patient for several months prior to clinical manifestation of TB. In contrast, analysis of responses to Ag60 (Saltini C. et al., 1993, Am Rev Respir Dis 145:1409-1414; van der Werf, T. S. et al., 1992. Med Microbiol Immunol 181:71-76) and Ag85B (McDonough, J. A. et al., 1992, J. Lab. Clin. Med. 120:318-322) failed to detect antibodies in these patients.
Hence, there is a particular need in the art for methods to detect TB infections at early stages in HIV patients since they comprise one of the largest populations at risk for TB throughout the world.
Antibodies in Urine
A number of laboratories have reported on antibodies, mainly to infectious agents, in urine. For example, Takahashi S; et al. (Clin Diagn Lab Immunol, 1998, 5:24-27) found antibodies to rubella virus in urine and serum samples from healthy individuals who underwent rubella vaccination. Shutov A M et al. Arkh (RUSSIA) 1996, 68:35-37 detected antibodies in urine to the virus causing hemorrhagic fever with renal syndrome (HFRS) and concluded that detection of antibodies to the virus both in the blood and urine can be used for earlier diagnosis Vereta L A; et al. (Vopr Virusol (RUSSIA) 1993, 38:18-21) used a commercial diagnostic indirect immunofluorescence assay to detection antibodies to the hantavirus in the urine of patients with HFRS. Koopmans M et al. (J Med Virol, 1995, 46:321-328) demonstrated presence of antibodies to human cytomegalovirus (HCMV) in urine samples by ELISA and immunoblot. Zhang X et al. (J Med Virol, 1994, 44:187-191) used commercial immunoassays to detect antibodies to hepatitis C virus (HCV) in urine. The same group (Constantine N T et al., Am J Clin Pathol, 1994, 101:157-161) detected antibodies to HIV in urine. Perry K R et al., Med Virol 1992, 38:265-270, detected IgG and IgM antibodies to hepatitis A and hepatitis B core antigens in urine specimens.
A group of Japanese investigates (Hashida S et al., J Clin Lab Anal, 1994, 8:237-246; Hashinaka K et al., J Clin Microbiol 1994, 32:819-22; Hashida S et al., J Clin Lab Anal 1994, 8:149-156 Hashida S et al., J Clin Lab Anal 1994, 8:86-95) diagnosed HIV-1 infection in asymptomatic carriers by detecting IgG antibody to HIV-1 in urine using an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) with recombinant proteins as antigen. They reported that sensitivity could be improved by a longer assay of bound enzyme activity by using concentrated urine samples and by the combined use of three different recombinant HIV antigens.
Urnovitz H B et al., (Lancet Dec. 11, 1993, 342:1458-9), discovered that 7 individuals who were negative for HIV-1 antibody in a licensed serum EIA were positive in a urine EIA and western blot (WB). Connell J A et al., J Med Virol 1993, 41:159-64, described a rapid, simple, and robust IgG-capture enzyme-linked immunosorbent assay (GACELISA) suitable for the detection of anti-HIV 1 and 2 antibodies in saliva and urine. An earlier study from this laboratory (Connell J A et al., Lancet, 1990, 335:1366-1369) described anti-HIV antibodies in urine by GACELISA). Gershy-Damet G M et al. Trans R Soc Trop Med Hyg 1992, 86:670-671, used these assays successfully for urinary diagnosis of HIV-1 and HIV-2 in Africa, using unprocessed saliva and urine specimens. They found the assay to be as accurate as conventional EIAs on serum tested under similar.
Dr. A. Friedman-Kien and his colleagues have examined paired urine and serum samples in a search for antibodies to hepatitis B surface antigen (HBs), hepatitis B core antigen (HBc), CMV and HIV in paired urine and serum samples from the same HIV-infected individuals (Cao Y et al., 1989, AIDS. Res. Hum. Retrovir. 5:311). In all individuals with anti-HIV antibodies in serum, anti-HIV antibodies were found in their urine; no such correlation was observed for HBs and CMV antibodies. The anti-HIV urine antibodies were of the IgG class, and gp160 and gp120 were the most consistently recognized proteins. Based on these observations, a urine based diagnostic assay for HIV-1 was developed.
In view of the prevalence of TB in the HIV-infected individuals, especially in the developing countries, and the risks and costs involved in collection of blood/serum for serodiagnosis, the present inventors evaluated the urine of TB patients for presence of anti-mycobacterial antibodies. They reasoned that since Mtb infects the mucosal surfaces in the lung, it may induce antibodies in mucosal tissues resulting in the presence of antibodies in the urine. The positive results of these studies are presented below. The ability to use urine as the sample material will make the test extremely attractive to public health officials and to industry.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.